生物化學(xué)：信號轉(zhuǎn)導(dǎo)通路

1Signal-TransductionPathways

信號轉(zhuǎn)導(dǎo)通路

Howcouldtheinsideofthecellknow whatwashappeningontheoutside?腦：神經(jīng)信號警告身體各部位，釋放激素激活腎上腺眼：瞳孔放大，視野變窄心臟：心率加速肺：氣管擴(kuò)張，呼吸頻率加快肌肉：血量增加，肌肉收縮肝臟：糖原分解，糖被釋放到血液脂肪細(xì)胞：脂肪酸被釋放到血液腸胃：流入消化系統(tǒng)的血量減少Timetoflee!髓質(zhì)皮質(zhì)醇腎上腺素去甲腎上腺素皮質(zhì)3干細(xì)胞分化：根據(jù)組織局部微環(huán)境的差異而分化成相應(yīng)的細(xì)胞2011年，Science評出

本世紀(jì)前十年十大科學(xué)成就：5.細(xì)胞重編程：將充分發(fā)育的細(xì)胞進(jìn)行“重編程”，使其成為多能干細(xì)胞-具有成為其身體中任何類型細(xì)胞的能力。Howcouldtheinsideofthecellknowwhatwashappeningontheoutside?signaltransduction!RobertJ.LefkowitzBrianK.Kobilka“forstudiesofG-protein-coupledreceptors”表彰他們對G蛋白耦聯(lián)受體的研究人高度緊張時“腎上腺素開始大量分泌”2012年，諾貝爾化學(xué)獎Forthediscoverythatmaturecellscanbereprogrammedtobecomepluripotent“發(fā)現(xiàn)成熟細(xì)胞可以重新編程而獲得多能性”ShinyaYamanakaJohnB.Gurdon2012年，諾貝爾生理/醫(yī)學(xué)獎7信號

受體

反應(yīng):手觸摸就是刺激(信號)，小葉合攏就是反應(yīng)。偶聯(lián)刺激到反應(yīng)之間的生化和分子途徑,就是這個反應(yīng)的信號轉(zhuǎn)導(dǎo)通路

觸摸含羞草后,小葉合攏8SignalTransductionPathway:Complicated9細(xì)胞信號轉(zhuǎn)導(dǎo)網(wǎng)絡(luò)的簡單模式（信號輸入）（信號輸出）10ImportantrolesofbiosignalingFunctionalintegrationofdistantorgans,tissuesandcellsrequirescommunication;Signalingisperhapsaprimalrequirementtorespondtoourenvironment;Thefoundationofanycomplexresponsepathwaylieswithcellularbiochemicals.BiosignalingIntercellular（細(xì)胞間）&Intracellular（細(xì)胞內(nèi)）11常見四種類型：Endocrine(內(nèi)分泌）Paracrine(旁分泌）Autocrine(自分泌）MembraneattachedproteinIntercellularsignaling

（細(xì)胞間信號）12Fourschemesofintercellularsignaling(1)13Fourschemesofintercellularsignaling(2)14Intracellularsignaling

（細(xì)胞內(nèi)信號）15Electron-micrographofmacrophage(pink)attackingEscherichiacoli(green)16M

吞噬處理入侵細(xì)菌及提呈抗原的機(jī)制17FcR

CR3Ca++srcPI3kPKCMAPKRhoGTPasegelsolinArp2/3PLCPLDActinrearrangementPhagocytosis;OxidativeactivationSignalsforphagocytosisSignalReceptorAmplificationTransductionResponsessecondmessengers信號轉(zhuǎn)導(dǎo)要素：信號或配體,受體,信號放大(產(chǎn)生第二信使),應(yīng)答和反饋調(diào)節(jié)18PARTⅠ1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPARTⅡ1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways191Fourbasiccharacteristics:1.1Specificity1.2Amplification1.3Desensitization/Adaptation1.4Integration20SpecificitySignalmoleculefitsbindingsiteonitscomplementaryreceptor;othersignalsdonotfit.thrombin凝血酶21oftenshort-lived&lowconcentration22Desensitization/Adaptation

ReceptoractivationtriggersafeedbackcircuitthatshutsoffthereceptororremovesitfromthecellsurfaceProducearapidandmajorcellularresponsetoatransientsignal23IntegrationWhentwosignalshaveoppositeeffectsonametaboliccharacteristicsuchasconcentrationofasecondmessengerX,orthemembranepotentialVm,theregulatoryoutcomeresultsfromtheintegratedinputfrombothreceptors242.Fourtypesofsignaltransducers2.1GatedIonChannels

2.1.1Ligand-gatedionchannels 2.1.2Voltage-gatedionchannels2.2ReceptorEnzymes2.3GProtein–CoupledReceptors andSecondMessengers2.4SteroidReceptors25Fourgeneraltypesofsignaltransducers26WhyIonChannels?Restingpotential:Asymmetricion-distributionActingpotentialGatedIonChannels Ligand-gatedionchannels Voltage-gatedionchannels27Restingpotential28IonConc.inMammalianCellsandSerum(mM）IonCytoplasmBloodSerumK+1404Na+12145Cl-4116proteincharges1389Mg+20.81.5Ca+2<0.00021.8WhyIonChannels:asymmetricion-distribution29asymmetricion-distributionRestingpotential30Actingpotential31ActingpotentialVoltage-gatedNa+channels&K+channels32pumpandionleakchannelsCl

-leakchannel332.1.1Ligand-gatedionchannel:Bindingofsomesmallmoleculeforcesanallosterictransitioninprotein,open/closechannel.

acetylcholine(乙酰膽堿)receptorionchannel2.1.2Voltage-gatedionchannelAchargedproteindomainmovesrelativetothemembraneinresponsetoachangeintransmembraneelectricalpotential.

(voltage-gatedNa+，

Ca2+，K+channels)34乙酰膽堿受體離子通道1AchACh35BindingofAChtoRcauseconformationalchange.AsM2helicestwistslightly,theLeuresidues(yellow)rotateawayfromthechannelandarereplacedbysmallerpolarresidues(blue).Thisgatingmechanismopenschannel,allowingpassageofCa,Na,orK乙酰膽堿受體離子通道2Closed Open36Voltage-gatedNa+channels137Voltage-gatedNa+channels2382.Fourtypesofsignaltransducers2.1GatedIonChannels

2.1.1Ligand-gatedionchannels 2.1.2Voltage-gatedionchannels2.2ReceptorEnzymes2.3GProtein–CoupledReceptors andSecondMessengers2.4SteroidReceptors39

2.2ReceptorenzymesAligand-bindingdomain(胞外)andanenzymeactivesiteoncytosolicside,connectedbyasingletransmembranesegment.CommonlyakinasethatphosphorylatesTyrresiduesinspecifictargetproteins（insulinreceptor）Other:synthesizethei.c.secondmessengercGMPinresponsetoex.c.signals

(thereceptorforatrialnatriureticfactor)40Activationofreceptortyrosinekinases41InsulinreceptortyrosinekinaseInsulinstructure42Insulinreceptorbindsinsulinandundergoesautophosphorylationonitscarboxyl-terminalTyrresidues.InsulinreceptorphosphorylatesIRS-1onitsTyrresidues.SH2domainofGrb2bindstoP–TyrofIRS-1.SosbindstoGrb2,thentoRas,causingGDPreleaseandGTPbindingtoRas.ActivatedRasbindsandactivatesRaf-1.Raf-1phosphorylatesMEKontwoSerresidues,activatingit.MEKphosphorylatesERKonaThr&aTyrresidue,activatingit.ERKmovesintothenucleusandphosphorylatesNucleartranscriptionfactorssuchasElk1,activatingthem.PhosphorylatedElk1joinsSRFtostimulatethetranscriptionandtranslationofasetofgenesneededforcelldivision.43Activationofglycogensynthasebyinsulin44Regulationofbloodglucoselevel45Oncogene-encodeddefectiveEGFreceptor462.Fourtypesofsignaltransducers2.1GatedIonChannels

2.1.1Ligand-gatedionchannels 2.1.2Voltage-gatedionchannels2.2ReceptorEnzymes2.3GProtein–CoupledReceptors andSecondMessengers2.4SteroidReceptors472.3GPCRandSecondmessengersThreeessentialcomponents:1.aplasmamembranereceptorwithseventransmembranehelicalsegments2.anenzymeintheplasmamembranethatgeneratesanintracellular2ndmessenger3.aguanosinenucleotide–bindingprotein(Gprotein)GPCR：感受物化刺激，包括多種神經(jīng)遞質(zhì)、肽類激素和趨化因子受體；超過半數(shù)的現(xiàn)代藥物靶向GPCR48NobelPrizeinPhysiologyandMedicine1994"fortheirdiscoveryofG-proteinsandtheroleoftheseproteinsinsignaltransductionincells"AlfredG.Gilman

1941-MartinRodbell

1925-199849ThreeessentialcomponentsofGProtein–CoupledReceptors5051AproteinbindsGuaninenucleotides(GDP,GTP);activatedinGTP-form,inactivatedinGDP-formIntegralmembraneprotein,heterotrimers(

);Havesimilar&subunits,butdifferin-subunitWhenG-proteinisactivated,thesubunitdissociatestointeractwithanenzymesthatgeneratesecondmessengers(e.g.cAMP)Others:smallG-proteins(~20-25kDa),e.g.Ras,Rho,Rac,etc,arenotmembranebound.Gprotein(GTP-bindingprotein)52Gprotein(discovery)M.Rodbell:atransducerprovidedthelinkbetweenreceptorandamplifier.A.G.Gilman:identify&purifytheGprotein.System:MutatedlymphomacellscontaininganormalreceptorandcAMP-generatingenzyme,wasyetunabletorespond(producecAMP),sinceitlackedthetransducermutatedcellnormalcell53“ON-OFF”switchisregulatedbyGTPorGDPboundform.AllG-proteinshasintrinsicGTPaseactivity,releasePiandinactivated.Activation:releaseofGDPandreplacedbyGTP54TheassociationofactiveGs

withadenylylcyclasestimulatesthecyclasetocatalyzecAMPsynthesisAdenosine3’,5’-cyclicmonophosphate(cAMP)55Taste-sensitive

areasoftongueFiveprimarytastes:BitterSweetSaltSourUmami

(Glutamate,delicious)TasteBack56Salty&sour:directlyviamembraneionchannels

Salt:detectedbypassagethoughsodiumchannelsSour:resultsfromtheeffectsofH+onvariouschannels.Umami:byamodifiedformofonebrainreceptorthatrespondstoglutamateasa

neurotransmitterDiversePathwaysDetectTastes(II)57Transductionforsweettastants58Twomajorsystems:2.3.1THEPKASYSTEM (cAMPasthesecondmessenger)

The-AdrenergicReceptorSystem2.3.2THEPKCSYSTEM

(DAG,IP3andCa2+asthesecondmessengers)RobertJ.LefkowitzBrianK.Kobilka“forstudiesofG-protein-coupledreceptors”表彰他們對G蛋白耦聯(lián)受體的研究人高度緊張時“腎上腺素開始大量分泌”2012年，諾貝爾化學(xué)獎6061G蛋白耦聯(lián)受體：感受物化刺激，包括多種神經(jīng)遞質(zhì)、肽類激素和趨化因子受體超過半數(shù)的現(xiàn)代藥物靶向62synthesizedinadrenalmedulla;belongstocatecholamines(兒茶酚胺）;targetcellsincludeliver,skeletalmuscle,heartmuscleandadipose;releasedinresponsetoacutestressEpinephrine腎上腺素signal63Epinephrine腎上腺素signalingpathwaycAMP64Epinephrine腎上腺素signalingpathway(2)65ActivationofcAMP-dependentproteinkinase(PKA)InactivePKA:Regulatory(R)subunits:auto-inhibitorydomainsburiedcatalytic(C)subunits:substrate-bindingsitesblockedbyRsubunitsRsubunits:autoinhibitorydomainsburiedActivePKACsubunitsopensubstratebindingsites66Epinephrinetriggersaseriesofreactionsinhepatocytesinwhichcatalystsactivatecatalysts,resultingingreat“amplification”ofthesignalx分子10,000x分子67PKAregulatesanumberofenzymesTheproteinsphosohorylatedbyPKAsharearegionofsequencesimilarityaroundtheSerorThrresiduethatundergoesphosphorylation,asequencethatmarksthemforregulationbyPKA.ThecatalyticsiteofPKAinteractswithseveralresiduesneartheThrorSerresidueinthetargetprotein,whichdefinethesubstratespecificity.6869霍亂毒素選擇性的催化Gsα亞基上的Arg201核糖化，使GTP酶活性喪失，不能將GTP水解成GDP，從而使Gsα處于不可逆激活狀態(tài),不斷刺激AC生成cAMP,胞漿中的cAMP含量可增加至正常的100倍以上，導(dǎo)致小腸上皮細(xì)胞膜蛋白構(gòu)型改變，大量Cl-和水分子持續(xù)轉(zhuǎn)運(yùn)入腸腔，引起嚴(yán)重腹瀉和脫水。70腸腔GsCT霍亂(Cholera)ACcAMP↑↑↑Cl-H2ONa+CT：CholeraToxin71DesensitizationofthePKAsystem1desensitizingβ-AdrenergicReceptor2degradingthesecondmessenger72GsbgrecruitsbARKtothemembrane,whereitphospho-

SerattheC-terminusofRecpt.barrbindstothepi-C-terminusofRecpt.Receptor-arrestincomplexentersthecellbyendocytosis.73β-ArrestinuncouplesreceptorfromitsGproteinandbringstogether3enzymesofMAPKcascade.(Onestimulustriggerstwodistinctpathways:thepathactivatedbyGproteinandMAPKcascade)74degradingthesecondmessenger75RegulationoftranscriptionbysteroidhormonesSteroidreceptor76PARTⅠ1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPARTⅡ1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways77Biosignaling:Howaretheyregulated?78IntegrationWhentwosignalshaveoppositeeffectsonametaboliccharacteristicsuchasconcentrationofasecondmessengerX,orthemembranepotentialVm,theregulatoryoutcomeresultsfromtheintegratedinputfrombothreceptors79SopEPKB/AktRac,CDC42PTKPI-3K

R調(diào)理后吞噬病原侵襲死亡信號生存信號細(xì)胞凋亡和粘膜屏障損壞細(xì)胞存活細(xì)胞凋亡是由：細(xì)胞內(nèi)“死亡/生存”信號之間的精密平衡來決定干擾該平衡就可改變病原對細(xì)胞凋亡的最終影響病原侵襲和吞噬對巨噬細(xì)胞凋亡的影響及其機(jī)制80細(xì)胞存活細(xì)胞凋亡Thebalancebetweenpro-andanti-apoptoticgenes/signalsdeterminethecellfate細(xì)胞接受到“死亡信號”，不一定就會死亡若同時也接受到“生存信號”，就可繼續(xù)存活81RegulatorymechanismsofBio-signals3.1Phosphorylationasaregulatorymechanism3.2Regulationoftranscriptionbysteroidhormones3.3Regulationofthecellcyclebyproteinkinases82RegulationofSignalingPathwaysExternalsignalsSecondmessengersModulatorproteinsFunctionTargetproteinsHormonescAMPOdorants

cGMPDrugsCa2+LightDAG IP3Mitogenichormones TyrosineGrowth kinasesfactorsproteinkinaseAndphosphatasestructuralproteinsAndenzymesmetabolicorphysiologicalresponsesPlasmamembrane83NobelPrizeinPhysiologyandMedicine1992EldwinG.KrebsEdmondH.Fischer

USA

“Reversibleproteinphosphorylationasabiologicalregulatorymechanism"J.Biol.Chem.216:121-132,1955Page84ProteinPhosphorylationNobelPrize1992NobelPrize2000EdmondH.FischerandEdwinG.Krebs

J.Biol.Chem.216:121-132,1955ArvidCarlsson,PaulGreengardandEricKandel85ProteinKinases1:

GPCRs

(5%),

2:

Kinases

(2.8%genome)

~25%

ofthemammalianintracellularproteinsundergoesreversiblephosphorylation86Proteinkinases(534humankinases)Ser/thrKinasescomprise80%ofallproteinkinasesAGC87Phosphorylation&de-phosphorylationarethemostcommonregulatorymechanisms,mediatedbyproteinkinaseandphosphatase,respectively

蛋白激酶

nNTPnNDP蛋白質(zhì)蛋白質(zhì)-nPi

nPi 蛋白磷酸酶

H2O88蛋白激酶（proteinkinase，PK）

植物和酵母中～2%的基因編碼蛋白激酶；而在哺乳動物中，高達(dá)5－8％。

根據(jù)磷酸化靶蛋白的氨基酸殘基的種類不同，蛋白激酶有絲氨酸/蘇氨酸激酶、酪氨酸激酶和組氨酸激酶等三類。部分蛋白激酶具有雙重底物特異性，既可使絲氨酸或蘇氨酸殘基磷酸化，又可使酪氨酸殘基磷酸化89CalciumIsaSecondMessengerinManySignalTransductionsNormally,[Ca2]iis~100nM(而細(xì)胞外：>1mM)Hormonal,neural,orotherstimulicauseeitheraninfluxofCa2+intothecellthroughspecificCa2+channelsintheplasmamembraneorthereleaseofCa2+fromERormitochondria,Changesin[Ca2]iaredetectedbyCa2+-bindingproteinsthatregulateavarietyofCa2-dependentenzymes-Calmodulin(CaM)90SHCGrb2GEFRas-GTPFRas-GDPFPiGAPSHCGrb2GEFRas-GTPFRas-GDPFPiGAPRas蛋白的上游和下游信號通路91PI3K－AKT途徑對細(xì)胞生存的調(diào)控機(jī)制92Mostofthesecomplicatedsignalingpathwaysaretransducedbythephosphorylationanddephosphoralationofsignalingprotein,regulatedbytheproteinkinasesandphosphatses93蛋白磷酸酶proteinphosphatase，PP蛋白磷酸酶的分類與蛋白激酶相對應(yīng)，分為絲氨酸/蘇氨酸型蛋白磷酸酶和酪氨酸型蛋白磷酸酶。有些酶具有雙重底物特異性對蛋白磷酸酶的研究還不如蛋白激酶那樣深入。但兩者的協(xié)同作用在細(xì)胞信號轉(zhuǎn)導(dǎo)中的作用是不言而喻的。

94Mechanismsusedbyhormones,retinoids,andVit.DtoregulategeneexpressionHormone(H),carriedtothetargettissueonserumbindingprot,diffusesacrossthePMandbindstoitsspecificreceptorprotein(Rec)inthenucleus.HbindingchangestheconformationofRec;formshomoorheterodimersandbindstospecificRregionscalledhormoneresponseelements(HREs)intheDNAadjacenttospecificgenesBindingregulatestranscriptionoftheadjacentgene(s),increasingordecreasingtherateofmRNAformation.Alteredlevelsofthehormoneregulatedgeneproductproducethecellularresponsetothehormone95Summary

FourbasiccharactersofbiosignalsFourschemesofintercellularsignalingFourgeneraltypesofsignaltransducers

gatedionchannels;receptorenzymes;G-proteincoupledreceptors；SteroidReceptorsGproteins:Heterotrimeric&monomeric; Activationanddeactivationmechanisms96Majorsecondmessengers:cAMP,IP3,DAG,Ca2+,cGMP.Usinganexampletoexplainthecascadeofevents:asinglemoleculeofhormoneactivatescatalystthatinturnactivatesanothercatalyst,andsoon,resultsinsignalamplificationandcellresponse97

細(xì)胞信號轉(zhuǎn)導(dǎo)調(diào)控與疾病防治Regulationofcellularsignalsinprevention&treatmentofdiseases以信號轉(zhuǎn)導(dǎo)蛋白為靶分子對疾病進(jìn)行防治STI571asaparadigm(范例）

forcancertherapyIn1960,describedthepresenceofaconsistentchromosomalabnormalityinCML（一種白血病)，so-calledPhiladelphiac